Efficient carbon and nitrogen transfer from marine diatom aggregates to colonizing bacterial groups

Bacterial degradation of sinking diatom aggregates is key for the availability of organic matter in the deep-ocean. Yet, little is known about the impact of aggregate colonization by different bacterial taxa on organic carbon and nutrient cycling within aggregates. Here, we tracked the carbon (C) and nitrogen (N) transfer from the diatom Leptocylindrus danicus to different environmental bacterial groups using a combination of 13C and 15N isotope incubation (incubated for 72 h), CARD-FISH and nanoSIMS single-cell analysis. Pseudoalteromonas bacterial group was the first colonizing diatom-aggregates, succeeded by the Alteromonas group. Within aggregates, diatom-attached bacteria were considerably more enriched in 13C and 15N than non-attached bacteria. Isotopic mass balance budget indicates that both groups showed comparable levels of diatom C in their biomass, accounting for 19 ± 7% and 15 ± 11%, respectively. In contrast to C, bacteria of the Alteromonas groups showed significantly higher levels of N derived from diatoms (77 ± 28%) than Pseudoalteromonas (47 ± 17%), suggesting a competitive advantage for Alteromonas in the N-limiting environments of the deep-sea. Our results imply that bacterial succession within diatom aggregates may largely impact taxa-specific C and N uptake, which may have important consequences for the quantity and quality of organic matter exported to the deep ocean.


Scientific Reports
| (2022) 12:14949 | https://doi.org/10.1038/s41598-022-18915-0 www.nature.com/scientificreports/ The ability of microbes to detect and exploit sinking diatom aggregates largely determines the efficiency of particle colonization [15][16][17] . Generally, heterotrophic bacterial taxa colonizing the aggregates are more diverse than free-living cells [18][19][20][21] and have higher metabolic activities than their free-living counterparts 6,10,22 . In particular, Flavobacteria and some Gammaproteobacteria (including Alteromonas and Pseudoalteromonas groups) due to their chemotactic and attachment abilities 23,24 are prominent bacterial groups found in association with marine particles 15,[24][25][26][27][28] , with an important contribution to aggregate degradation in the dark ocean 29 . In recent studies 20,30 , it has been shown that colonization of sinking particles may act as a vector inoculating viable cells into the meso-and bathypelagic zones, with a potentially large impact on the microbial community composition of the deep sea 30 . Yet, our understanding of the importance and role of different bacterial groups in the C and N cycling within the aggregates and, hence, determining the amount of C and N exported to the surrounding water is still limited.
We combined stable isotope probing with taxonomic identification techniques aiming to analyze the dynamics in diatom aggregate colonization by different phylogenetic groups of bacteria, as well as their use of diatomderived C (DDC) and N (DDN). We performed roller tank incubation experiments (72 h) by inoculating a 13 C and 15 N pre-labelled aggregate-forming diatom culture (Leptocylindrus danicus) with natural seawater samples containing diverse bacterial communities. We followed bacterial aggregate colonization and use of DDC and DDN using single-cell analyses based on Catalyzed Reported Deposition-Fluorescence In Situ Hybridization (CARD-FISH) and nano-scale Secondary-Ion Mass Spectrometry (nanoSIMS). Our data show that diatom colonization was mostly dominated by two Gammaproteobacteria groups, Pseudoalteromonas and Alteromonas, which followed a distinct succession and DDC/DDN incorporation pattern, suggesting a specific role of each taxonomic group in the colonization and degradation of marine aggregates.

Results
Dynamics of bacteria within the aggregates. A 13 C and 15 N pre-labelled L. danicus culture was inoculated with an environmental non-labelled microbial community collected at 50 m water depth in the Atlantic Ocean (Cape Blanc, Mauretania). Aggregation experiment was done in roller tanks. In this work, we differentiate between bacteria that were physically attached to the diatom cells from those that were found within the aggregates (and hence were not free-living), but not directly attached to diatoms (here on called non-attached). An example of the visual identification of both bacterial fractions is shown in Supplementary Fig. S1. Both, attached and non-attached fractions were detected by CARD-FISH hybridization and counted manually by epifluorescence microscopy analysis. At the start of the experiment, we detected only a few bacteria (< 0.1 bacteria diatom −1 ) associated to the initially small diatom aggregates (< 0.5 mm), among which 23% of the bacteria were directly attached to the diatoms (Fig. 1). Macroaggregates (> 1 mm) were formed a few hours (< 3 h) after the initiation of the experiment. During the experiment, bacterial abundances within the formed aggregates increased, reaching to > 1 bacterium diatom −1 after 30 h and stabilized around this value until the end of the experiment (72 h). Due to the heterogeneous nature of diatom aggregates, the bacterial abundances were highly variable (coefficient of variation of 84% on average, ranging between 21 and 143%).
At the start of the experiment, only Alphaproteobacteria and Bacteroidetes were associated with L. danicus within the aggregates, although at low abundances (< 0.05 cells diatoms −1 , Fig. 1, Table S1). The abundances of these groups stayed below 0.2 cells diatom −1 during the entire experiment (72 h, Fig. 1). The Gammaproteobacteria Alteromonas and Pseudoalteromonas were not detected at the beginning of the experiment but showed a fast accumulation within the aggregates over time, reaching up to 0.6 and 1.1 cells diatom −1 , respectively. As a result, the bacterial community shifted from Bacteroidetes and Alphaproteobacteria dominance toward a Gammaproteobacteria dominance. Overall, the proportion of cells attached vs. non-attached diatoms (bacteria that are within the aggregates but with no physical contact with the diatom cells) to diatoms was significantly higher (p < 0.01, Wilcoxon test) for Pseudoalteromonas (51%) and Alteromonas (32%) than for Alphaproteobacteria (14%) and Bacteroidetes (4%) (Fig. 2).
Phytoplankton and bacterial cell-specific isotope enrichment. To analyze the transfer of 13 C and 15 N from pre-labelled diatom aggregates to bacteria, a total of 407 diatom cells and 213 bacteria were quantified at single-cell level using nanoSIMS. For the nanoSIMS analysis, we only focused on Alteromonas and Pseudoalteromonas groups, as both groups represented most of the aggregate-associated bacterial cells as observed in Fig. 1. The high number of cells analyzed allowed us to calculate the proportion of diatom-derived carbon (DDC) and nitrogen (DDN) in individual bacterial cells. At the beginning of the experiment, diatom fractional isotope abundances for C and N (A 13 C and A 15 N, respectively) averaged 2.3 ± 0.1 and 66.4 ± 6.2 atom% (Fig. 3A,C) but decreased linearly to 1.9 ± 0.41 and 54.4 ± 21.6 atom%, respectively, after 72 h. Considering the low natural fractional isotopic abundance of 13 C (1.08 atom%) and 15 N (0.37 atom%), this corresponded to a decrease of 35% and 18% of their isotopic enrichment, respectively. Significant 13 C and 15 N isotopic enrichments were also detected in bacterial cells. While Alteromonas showed higher A 15 15 N between both bacterial groups was highest at the beginning of the incubation (twofold) and tended to be reduced towards the end of the incubation (1.2-fold, Fig. 3). Bacterial cells of both groups were on average more enriched in 13 (Fig. 3). Using a mass balance approach, we calculated that DDC and DDN represented on average 17% (1-34%) and 63% (31-122%) of the aggregate-embedded bacterial C and N biomass, respectively (Fig. 4). The DDC was similar for both strains, whereas DDN was higher for Alteromonas than for Pseudoalteromonas (Fig. 4, Table S2). The low DDC:DDN ratio for both Pseudoalteromonas and Alteromonas indicated a preferential diatom-derived N uptake for both groups, particularly of Alteromonas, which showed an average DDC:DDN ratio consistently lower than Pseudoalteromonas (0.18 and 0.43, respectively).

Discussion
Heterotrophic bacteria living in association with sinking marine aggregates hydrolyze particulate organic matter (POM) to dissolved organic matter (DOM), releasing it to the surrounding water. Combined, bacterial hydrolyzation and respiration of organic matter cause the attenuation of the export of organic matter to deeper depths, lowering the efficiency of the biological carbon pump 13 . Previous studies have shown that attached heterotrophic bacteria drive C and N turnover 31,32 and the breakdown of aggregates through enzymatic hydrolysis 33,34 . Yet, due to the small spatial scale in which these processes occur, bulk analysis cannot accurately determine the importance of these diatom-bacteria associations on C and N cycling within aggregates vs. those in the surrounding water (so-called free-living bacteria). In this work, we have coupled nanoSIMS and CARD-FISH single-cell analysis, to assess the role of phylogenetically different bacteria in the colonization of diatom cells and in the C and N cycling within diatom aggregates. Here, we differentiate between attached cells (the ones that are in physical contact with the diatoms) from the non-attached ones (bacteria within the aggregates but with no physical contact with the diatom cells). In contrast to free-living bacteria, the non-attached cells are found within the aggregates, most likely embedded within the organic matrix of the aggregates such as transparent exopolymer particles (see Figs. S1, S3). Using a 15 N and 13 C pre-labelled axenic L. danicus culture, which is described as an important aggregate-forming diatom 4 , and fresh field-collected bacteria, we show the importance of a fast  www.nature.com/scientificreports/ aggregate colonization ability and the following bacterial succession for an efficient C and N assimilation within the aggregates. The increasing bacterial abundances on diatom aggregates observed during our roller tank incubations are in good agreement with previous reports [35][36][37] . Due to the rapid aggregate colonization by non-attached bacteria in the first hours of incubation 36 and the following high growth-efficiency of aggregate-associated bacteria observed at the early colonization phase 38 , it is likely that the increasing bacterial abundances resulted from a combination of colonization and active growth within the aggregates. For example, high attachment and subsequent colonization rates would explain the switch of Pseudoalteromonas abundance from the non-attached to the attached fraction between 39 and 48 h of incubation. On the other hand, the consistently higher abundance of Alteromonas in the attached fraction compared to non-attached and their high N uptake may suggest an active growth within the aggregates. Yet, aggregate colonization by the low abundant non-attached Alteromonas was also likely happening. Regardless of the process responsible for the increase in colonizer abundance of the attached fraction, we derived that the net bacterial abundance increase would be 6 d −1 during the first 30 h of incubation. This is one order of magnitude higher than most of the estimates for marine free-living bacteria (~ 0.2-0.4 d −139,40 ) but closer to the range of reported values for aggregate-attached bacteria (0.5 to > 4 d −17 ).
Bacterial colonization is mediated by the ability of bacteria to detect and exploit marine aggregates 41 . Both Alteromonas and Pseudoalteromonas dramatically increased in abundance within the diatom aggregates and are well known to colonize marine particles by chemotactic motility 24,42,43 . They have also been shown to efficiently use algal-derived organic matter 44,45 . Yet, such abilities have been more frequently described in other bacterial groups such as Flavobacteria or Roseobacter 15,46,47 , while in the current work, these groups were less frequently attached to the diatom aggregates than members of the Pseudoalteromonas and Alteromonas groups. The observed high variability in the composition of the aggregate-associated bacterial community may be related to the distinct microbial taxa harbored by different phytoplankton species 45,48 . Another possibility is that both groups have better adaptations to efficiently colonize diatom-aggregates, in particularly Pseudoalteromonas, Table 1. Isotopic abundances presented as fractions of identified bacteria (average ± standard deviation) including all time points. Attached bacteria are significantly more isotopically enriched than non-attached ones for both phylogroups and both isotopes (Wilcoxon-Mann-Whitney, p < 0.05).  www.nature.com/scientificreports/ whose physiological abilities to live attached to aggregates would provide a competitive advantage against other members of the aggregate-associated community 42 . The subsequent changes in attached community composition, with increasing Alteromonas abundance in the attached fraction after 39 h, could be related to myriad microbial interactions that happen in the aggregates, including viral lysis or bacterial competitive interactions 55 , that may control colonization succession. Bacteria growing within aggregates show a higher exoenzymatic activity compared to free-living bacteria, which facilitates the access to more recalcitrant DOM 1,49 . The fast colonization ability of Pseudoalteromonas also suggests that they may hold a key role during the initial phase of aggregate hydrolysis by exoenzymatic activity, facilitating the subsequent aggregate colonization by other microbial taxa. The micro-scale succession of colonizing taxa within marine aggregates has been previously described 35 , however, our study suggests that Pseudoalteromonas may be one of the first responsible for the diatom-aggregate breakdown, which may impact the C and N assimilated by other taxa.
Monitoring the isotopic composition of bacteria at different timepoints allowed us to further investigate the relative importance of colonization and in situ microbial growth. In stable isotope probing experiments, the isotopic ratio of the targeted organisms is expected to increase with time, until an isotopic equilibrium is reached (i.e., when the isotope ratio of targeted organisms equals that of the isotope source pool, see model in Fig. S2). We observed a considerable variability in the isotopic enrichment over time, with Alteromonas A 15 N as high as the diatoms A 15 N after 21 h but decreasing thereafter. The isotopic equilibrium reached within the first hours of the incubation demonstrated the efficient use of DDN by Alteromonas. The counterintuitive decrease in DDN between 30 and 72 h of the incubation could result from fast microbial turnover rates (with rates of bacterial colonization and detachment as fast as 3 h 50 ), which would prevent the observation of a continuous increase of their isotopic composition. However, it is also possible that besides the diatom derived 15 N, bacteria used alternative non-labelled sources of N. A recent study has shown that marine aggregates may constitute oxygen depleted microenvironments favorable for microbial N 2 fixation 51 . According to the authors, such microhabitats would need around 2 days to develop, which falls in good agreement with the decrease in A15N observed for Alteromonas and indicates that the Alteromonas group may have used the recently fixed and non-labelled N in the latest phase of the incubation. Despite the observed trends, our results suggest that Alteromonas group is able to take up a large fraction of DDN within a few hours of aggregate formation. Such fast N uptake ability may have an important impact on the N circulation and remineralization within the first stages of aggregate sinking, retaining a big fraction of the aggregate derived N in the surface ocean.
While Alteromonas represented most of the attached bacteria cells detected after 15 h of incubation, the relatively low DDN of Pseudoalteromonas (38% after 30 h) implies that alternative non-labelled N sources play a substantial role for the build-up of bacterial biomass. Although parts of this N uptake could derive from N-fixation, the time needed for the formation of an anoxic microenvironment within the aggregates 51 does not explain the initial low DDN. Thus, the relatively low DDN in Pseudoalteromonas may have been related to other sources rather than those derived from N-fixation, such as dissolved N incorporated from the seawater. We cannot disregard the impact of grazers and viral infections on the incorporation of non-labelled substrates, as bacterial lysis through grazing or virus activity could also recirculate dissolved organic substrates [52][53][54] . Despite the origin of both, non-labelled and 15 N-labelled substrates, our results clearly show distinct colonization strategies by Pseudoalteromonas and Alteromonas. Pseudoalteromonas may have incorporated a high amount of non-labelled N at the beginning of the incubation in order to gather enough energy for a rapid aggregate colonization. On the other hand, Alteromonas was more dependent on the aggregate-derived 15 N, delaying the aggregate colonization to a secondary phase.
In contrast to DDN, we found that cell-specific DDC incorporation rates were very similar for both analyzed bacterial groups. This consistent C uptake agrees with a previous study showing similar algal-derived dissolved organic C (DOC) uptake by two copiotrophic groups associated with the diatom phycosphere 47 , suggesting that C assimilation heterogeneity in aggregate-attached bacteria is relatively low and barely varies between different phylogenetic groups. Another interesting observation is that the cells of the attached fraction were able to incorporate substantially more C than the non-attached ones, while no difference was observed for N (Fig. 4). This result is in good agreement with previous observations where higher isotope uptake was observed in bacteria physically attached to phytoplankton cells than in their free-living counterparts 55 . Despite we did not target the free-living fraction in this study, current results further highlight that physical contact between bacteria and phytoplankton is the most effective way of nutrient transfer. Similar observation regarding close physical contact as a requirement for efficient nutrient transfer was shown for other types of interactions e.g. fungi-bacteria, where a proximity of 1.7 µm was necessary for efficient nutrient and water transfer from fungi to bacteria while bacterial cells further away could not receive the nutrients provided by the fungi mycelium, remaining metabolically inactive 56 .
The low C assimilation by both bacterial groups studied here was also remarkable, representing only 10-30% of DDC. According to our calculations (and considering an average of 1 bacteria per diatom), at this rate, total particle degradation would take 10 times longer than previous estimations (8-9 days 31 ). Such low C assimilation in comparison to N may be a consequence of different metabolic pathways for distinct compounds. While the majority of the incorporated N was assimilated as biomass, it is possible that a large fraction of DDC was metabolized and remineralized as CO 2 through bacterial respiration. In fact, high respiration rates have been measured in aggregates (with C turnover rates of 0.1-0.2 d −157 ) releasing most of the DDC back to the system as CO 2 ; microbial grazing and viral lysis would explain the low microbial abundance despite the potentially high respiration rates 32,54 . Another possibility for the difference observed between DDN and DDC is a preferential decomposition of particulate organic nitrogen (PON) as compared to carbon, as previously suggested 34,58 , which falls in good agreement with increasing C:N ratio with increasing depth, and is generally observed in sinking materials across the Oceans 59 . According to our results, N would be degraded twice as fast as C within www.nature.com/scientificreports/ the aggregates, meaning these latter would become more C-rich while sinking (also supported by previous observations 33 ). Thus, contrary to the initial hypothesis, bacterial degradation would change the stoichiometry of the sinking particles, marginally increasing the efficiency of the biological carbon pump. The current work highlights the importance of a fast colonization ability for an efficient C and N assimilation by bacteria within diatom aggregates. The fast bacterial turnover, together with the high uptake of diatom-derived substrates (mostly N) within the first 21 h of incubation, strongly indicate that the first colonization phase is a key step in C and N cycling between diatoms and associated heterotrophic bacteria. The imbalance that we observed between the use of DDN and DDC further highlights the importance of microbial succession within the aggregates on the C export to the deep ocean. However, how other factors, such as increasing pressure 60,61 and decreasing temperature 62 with increasing depth, may impact the C and N assimilation by aggregate-associated bacteria should be further addressed to understand the element cycling in the meso and bathypelagic realm. Nevertheless, our results represent an important step forward in determining the impact of cell-specific colonization on the N and C cycling within settling diatom aggregates.

Diatom cultures and incubation with stable isotopes.. Leptocylindrus danicus (strain CCMP470)
were cultured in a f/2 medium and grown at constant temperature 63 . The medium was prepared with 13 C-bicarbonate (NaH 13 CO 3 , Sigma Aldrich) and 15

Catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH). CARD-FISH
analysis was performed following the protocol from Pernthaler et al. 64 . To analyze microbial abundance, a small piece of the polycarbonate filters was cut. This filter piece contained several intact aggregates and their associated bacteria, which were used to determine the abundance of different microbial groups by CARD-FISH. The filter cuts were dipped in 0.1% low melting-point agarose to avoid detachment of the cells from the filter surface. . Bacterial cells were considered as attached when directly in contact with a diatom or in contact with another bacterium which was in contact with a diatom. CARD-FISH enumerations are usually expressed relative to DAPI stained bacterial cells. In our case, within diatom aggregates, the DNA fluorescence derived from DAPI staining of the large nuclei of diatom cells hindered the fluorescence of bacteria preventing exhaustive enumeration. Therefore, the total bacteria abundances were estimated relative to the hybridized cells using the mix of EUB I, II & III probes which has been demonstrated to detect > 99% of prokaryotic cells in the sampled region 66 In addition to epifluorescence microscopy, Helium Ion Microscopy (HIM, ZEISS) images were also taken. Area of interest containing attached bacterial cells were selected for HIM imaging prior to the nanoSIMS analysis. Analytical conditions employed during HIM imaging are depicted on the individual images. Sample preparation prior to HIM image involves chemical fixation by paraformaldehyde 1.5% at 4 °C for 24 h followed by repetitive washing step in PBS 1X followed by short dehydrations in 96% followed by air drying and storage at − 20 °C until imaging. Parallel filter pieces (metal coated 0.2 µm polycarbonate filters GTTP type, Merck, Millipore) containing diatom aggregates collected for the nanoSIMS analysis at various time points were selected for HIM imaging.

NanoSIMS analysis.
For the nanoSIMS analysis, additional cuts from the same polycarbonate filters used for the previous CARD-FISH analysis were collected. Prior the nanoSIMS, an additional CARD-FISH analysis was performed in order to locate the Alteromonas and Pseudoalteromonas cells that we were going to analyze with the nanoSIMS. Filters containing 15 N and 13 C labelled phytoplankton cells and bacteria were hybridized using the HRP-labeled ALT1413 and PSA189 oligonucleotide probes as described above. The amplification step was done using fluorine containing fluorescently labelled tyramides with Oregon Green® 488 (ThermoFisher, Germany) according to Musat et al. 71 . Diatom cells and hybridized bacteria were mapped using a laser microdissection system (LMD, Zeiss, Germany) equipped with an inverted epifluorescence microscope. The LMD marked areas were further analyzed correlatively using a nanoSIMS 50 L (Cameca, Gennevilliers, France). A 2 pA primary Cs + ion beam of 16 keV was focused into about 70 nm spot at the sample surface during the analysis. Samples were analyzed in a 512 × 512 pixel raster over 30 × 30 µm 2 areas with 5 ms dwell time per pixel. Before the analysis, the sample surface was treated with 10 nA of low-energy (50 eV) Cs + beam over 80 × 80 µm 2 area for 10 min. The secondary ions species were analyzed for their mass to charge ratio (m/z) and detected using the seven available collectors: 12 C − , 13 C − , 19 F − , 12 C 14 N − , 12 C 15 N − and 13 C 14 N − , 31 P − , 32 S − . Two secondary ion species ( 12 C 15 N − and 13 C 14 N − ) were detected by switching the deflector voltage in combined analysis mode. The mass resolving power (MRP) was checked to be between 7000 and 10,000 with the exit slit width of 70 µm, entrance slit width of 20 µm, aperture slit of 200 µm and with the energy slit cutting about 30% of secondary ions in high-energy tail of their energy distribution. Under these analysis conditions, we observed that cells were sputtered completely within 10 scans. Scans 3-8 were considered for the analysis employing LANS software 72 allowing for accumulation of the scans with lateral drift correction and quantitative analysis of isotopic ratios ( 13 C/ 12 C, 13 C 14 N/ 12 C 14 N and 12 C 15 N/ 12 C 14 N). To determine cell-specific isotope ratios, regions of interest (ROIs) were drawn inside the cells, avoiding the cell-surrounding areas.
Data presentation and statistical analyses. The C and N cellular isotope composition measured by nanoSIMS is presented as fractional isotope abundances (atom%): The addition of non-isotopically enriched C and N molecules as well as potential leakage of labelled smallmolecular-weight cellular components during various chemical steps of the CARD-FISH treatment leads to an isotopic dilution of the analyzed cells 73,74 . In order to correct for this effect, diatoms and bacterial cells were analyzed by nanoSIMS with and without CARD-FISH treatment. 13 C dilution factors were 2.5% and 29.7% and 15 N dilution factors were 6.7% and 53.4% for diatoms and bacteria respectively. All the fractional isotopes abundances presented here are corrected from CARD-FISH isotopic dilutions.
At each time point, the proportion of DDC and DDN in bacteria was calculated as follows: With A cell being the fractional isotopic abundance of bacteria cells at each time point and A t0 the mean fractional isotopic abundance in non-labelled samples. The isotopic dilution of diatoms throughout the experiment (see "Results" section) was accounted for by using the average fractional isotopic abundance.